Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, Norman, OK 73019-5251; rhcichewicz@ou.edu.

Abstract

Mass-spectrometry-based metabolomics and molecular phylogeny data were used to identify a metabolically prolific strain of Tolypocladium that was obtained from a deep-water Great Lakes sediment sample. An investigation of the isolate's secondary metabolome resulted in the purification of a 22-mer peptaibol, gichigamin A (1). This peptidic natural product exhibited an amino acid sequence including several β-alanines that occurred in a repeating ααβ motif, causing the compound to adopt a unique right-handed 311 helical structure. The unusual secondary structure of 1 was confirmed by spectroscopic approaches including solution NMR, electronic circular dichroism (ECD), and single-crystal X-ray diffraction analyses. Artificial and cell-based membrane permeability assays provided evidence that the unusual combination of structural features in gichigamins conferred on them an ability to penetrate the outer membranes of mammalian cells. Compound 1 exhibited potent in vitro cytotoxicity (GI50 0.55 ± 0.04 µM) and in vivo antitumor effects in a MIA PaCa-2 xenograft mouse model. While the primary mechanism of cytotoxicity for 1 was consistent with ion leakage, we found that it was also able to directly depolarize mitochondria. Semisynthetic modification of 1 provided several analogs, including a C-terminus-linked coumarin derivative (22) that exhibited appreciably increased potency (GI50 5.4 ± 0.1 nM), but lacked ion leakage capabilities associated with a majority of naturally occurring peptaibols such as alamethicin. Compound 22 was found to enter intact cells and induced cell death in a process that was preceded by mitochondrial depolarization.

Secondary metabolite profiles of Tolypocladium sp. T2. HPLC analysis (PDA scan at 200‒600 nm) of the secondary metabolite profiles of T2 grown in PDB medium (A) and PDB medium with 2 g/L NaNO3 (B). Isolated compounds are categorized in the dashed boxes according to their regions on the chromatograms. Structures of the secondary metabolites from T2 are shown in C and D. Additional structural information and detailed renderings of the compounds discussed in this report are provided in . Amino acid codes for the rare residues: BALA, beta-alanine; J, isovaline; PA, pipecolic acid; U, 2-aminoisobutyric acid.

Single-crystal X-ray diffraction of gichigamin A (1). (A) The complete structure of the gichigamin A monomer generated using PyMol. The C-terminal Gly-ol hydroxyl is displayed with the highest occupancy model (73%). (B) N-terminal α-β-motif of 1. Pro2 is shown coordinating through two hydrogen bonds to the i + 3 and i + 4 residues. (C) A perpendicular view of the unique 311-P-helical structure resulted from the inclusion of BALA residues in 1. The N- and C-termini were removed for clarity. (D) For the purpose of comparison, a canonical 310 helix is shown that was generated with Φ- and Ψ-angles of −49° and −29° for poly-Ala dodecamer using the PepMake 1.2 server at pepmake.wishartlab.com/.

Comparison of the biosynthetic clusters NPT1, NPT2, and NPTi following analysis by AntiSMASH v3.0.2 (fungismash.secondarymetabolites.org/#!/start). The numbers and codes appearing in this figure identify the isolates, contigs, and genes discussed in this article. The three putative NRPS-PKS clusters each contained 21 NRPS modules, as well as the signature of a PKS module [an acyltransferase (AT) and a ketosynthase (KS)].

Antitumor effects of 1 in a pancreatic tumor xenograft model. Athymic mice were bilaterally implanted with MIA PaCa-2 tumors and treated with 1 at a dose of 0.1 mg/kg on days 0 and 3 (open triangles) or 0.25 mg/kg on day 0 (open circles). The results represent the average change in tumor volume ±SE. *P < 0.05; **P < 0.01; ****P < 0.0001 compared with untreated control (filled squares) by a two-way ANOVA with a Dunnett’s posthoc test.

Effects of 1 and 22 on whole-cell patch-clamp electrophysiology and mitochondrial depolarization in PANC-1 cells. Shown are the effects of (A) 1 µM of 1 (n = 8) and (B) 0.025 μM of 22 (n = 8) on macroscopic whole-cell ionic currents elicited in individual representative PANC-1 cells. Control families of step-derived current recordings for A and B are in , respectively. (A) Ramps recorded without compound (a), upon application (b), and after 5-min (c) and 8-min (d) exposure to 1. Acute and 5-min ramps exhibited small additional increases in inward currents. The 5-min ramp showed larger outward currents compared with the acute ramp. Subsequent rapidly developing linear leak currents (8 min) resulted in cell loss (all studied cells were lost between 10 and 15 min). (B) Shown are the first 12 ramps recorded over 15-min encompassing control, acute, and 5-min exposure to 22. Not shown for clarity are stable ramps recorded over the subsequent 30 min before cell loss due to breakdown of recording configuration. Ramps exhibited no changes in magnitude and voltage-dependent properties, nor did leak currents develop over the 35-min recording. Membrane potential (U) is shown in millivolts on the left vertical axis, current magnitude (I) is shown in picoampere on the right vertical axis, and time duration (time) of voltage steps and elicited currents is shown in milliseconds on the horizontal axis. (C and D) PANC-1 cells were treated for 4 h with 1, 22, or alamethicin at the indicated concentrations. The extent of cell death, as determined by 7-AAD staining (C), and mitochondrial depolarization, as measured by incorporation of mitopotential dye (D), were quantified by flow cytometry. n = 3–5 independent experiments ±SEM ****p < 0.0001 by two-way ANOVA with Dunnett’s posthoc test compared with vehicle control.